Project Summary Neuropeptides are essential neuromodulators in the brain. They are released into the extrasynaptic space, where they diffuse over long distances and signal through G protein coupled neuropeptide receptors. Neuropeptides control cognition, sensorimotor processing, and energetics through changes in vascular tone and blood flow in the nervous system. Pharmacological and molecular genetic studies have implicated alterations in neuropeptide signaling as a contributor to brain dysfunctions, including migraines, addiction, motivation and stress. Although widely expressed in the brain, remarkably little is known about when and where neuropeptides are released. Monitoring the release of neuropeptides in real-time in awake animals performing complex behaviors would be transformative, enabling the elucidation of the function of neuropeptides in regulating neural circuits in the brain. In response to RFA-MH-16-775, we propose to develop and validate an innovative neurotechnique for optically measuring release of neuropeptides in a cell-specific and circuit-specific processes in the brain. The new technology is based on cell-based neurotransmitter fluorescent engineered reporters, referred to as CNiFERs, which were original developed for detecting the release of classical, small molecule neurotransmitters. A CNiFER is a clonal HEK293 cell that is engineered to express a specific G-protein coupled receptor and a genetically encoded fluorescence-based intracellular calcium sensor. CNiFERs are implanted in the brain, where they produce minimal inflammation and remain viable for days, and have been used successfully to measure volume transmission of dopamine, norepinephrine and acetylcholine in vivo during learning. Three neuropeptide CNiFERs will be developed and used for test-bed validation projects within our own laboratories: Orexin, which is important in sleep regulation as well as drug seeking and reinstatement, Somatostatin which has been implicated in depression, motivation and learning, and Vasoactive Intestinal Peptide, which as been implicated in neuroplasticity and learning. For collaborative projects, we will further construct four additional neuropeptide CNiFERs for detecting release of Dynorphin, Corticotropin-Releasing Factor, Neuropeptide Y and Substance P. Each neuropeptide CNiFER will be subjected to rigorous in vitro testing prior to their use to study the dynamics and consequences of release of neuropeptides in vivo.